The present invention relates to a novel method for producing a functional peptide nucleic acid monomer, a functional peptide nucleic acid oligomer produced by that method, and its intermediates. More particularly, the present invention relates to a production method comprising introducing one type or two or more types of a functional molecule post-synthetically following introduction of a precursor PIVA monomer unit into a PNA oligomer.
Nucleic acids consist of DNA and RNA that govern the genetic information of living organisms. In contrast, peptide nucleic acids (PNA) refers to modified nucleic acids in which the sugar phosphate skeleton of a nucleic acid has been converted to an N-(2-aminoethyl)glycine skeleton (FIG. 1). Although the sugar-phosphate skeletons of DNA/RNA are subjected to a negative charge under neutral conditions resulting in electrostatic repulsion between complementary chains, the backbone structure of PNA does not inherently have a charge. Therefore, there is no electrostatic repulsion. Consequently, PNA has a higher ability to form double strands as compared with conventional nucleic acids, and has a high ability to recognize base sequences. Moreover, since PNA is extremely stable with respect to nucleases and proteases in the living body and is not decomposed by them, studies are being conducted on its application to gene therapy as an antisense molecule.
As a result of using PNA in technology that conventionally used DNA as a medium, it has become possible to compensate for those shortcomings of DNA that were heretofore unable to be overcome. For example, PNA can be applied to xe2x80x9cDNA microarray technologyxe2x80x9d for rapid and large-volume systematic analysis of genetic information, as well as recently developed xe2x80x9cmolecular beaconsxe2x80x9d used a probes capable of detecting that a base sequence has been specifically recognized using emission of fluorescent light. Since both of these use DNA lacking enzyme resistance as the medium, strict sampling is required when using these technologies. The satisfying of this requirement is the key to achieving greater sophistication of these technologies.
On the other hand, since PNA is completely resistant to enzymes, by substituting the use of DNA for PNA in DNA microarray technology and molecular beacons, the previously mentioned technical shortcomings can be overcome, leading to expectations of being able to take further advantage of the merits of these technologies.
Although there are many other fields in which the use of PNA is expected to lead to further advancements in addition to DNA microarray technology and molecular beacons, in these fields it will be necessary to design novel PNA monomers by enabling PNA to function efficiently, namely by realizing the efficient introduction of functional molecules into PNA monomers.
Since ordinary solid-phase peptide synthesis methods are used for PNA oligomer synthesis methods, classification of PNA monomer units according to PNA backbone structure yields the two types consisting of Fmoc type PNA monomer units and tBoc type PNA monomer units (FIG. 2).
Methods for synthesizing Fmoc type PNA monomer units have already been established, and since their oligomer synthesis can be carried out using an ordinary DNA automated synthesizer, synthesis can be carried out on a small scale by the following route: 
(wherein X represents guanine, thymine, cytosine or adenine).
Initially, tBoc type PNA monomer units like those shown below: 
were used and this was followed by the establishment of more efficient synthesis methods. 
However, since the previously mentioned Fmoc type was developed that offered easier handling, the frequency of use of the tBoc type is decreasing.
However, when introducing a functional molecule other than the four types of nucleic acid bases of guanine, thymine, cytosine and adenine, such as when introducing a photofunctional molecule, there are many cases in which the functional molecule to be introduced is unstable under alkaline conditions, and thus a tBoc type of PNA backbone structure that is not used under alkaline conditions is highly useful. A patent application for a xe2x80x9cmethod for producing t-butoxycarbonyl-aminoethylamine and amino acid derivativesxe2x80x9d has already been made by the inventors of the present invention as Japanese Patent Application No. 2000-268638.
In addition, there are also five examples of synthesis of monomer units of photofunctional oligo PNA in the prior art. Although all of these use the above route, their yields are either not described or are extremely low (Peter E. Nielsen, Gerald Haaiman, Anne B. Eldrup PCT Int. Appl. (1998) WO 985295 A1 19981126, T. A. Tran, R.-H. Mattern, B. A. Morgan (1999) J. Pept. Res, 53, 134-145, Jesper Lohse et al. (1997) Bioconjugate Chem., 8, 503-509, Hans-georg BAtz, Henrik Frydenlund Hansen, et al. Pct Int. Appl. (1998) WO 9837232 A2 19980827, Bruce Armitage, Troels Koch, et al. (1998) Nucleic Acid Res., 26, 715-720). In addition, since the structures of the compounds used have the characteristic of being comparatively stable under alkaline conditions, they are expected to be uable to be produced in good yield using a method similar to the above-mentioned methods of the prior art, namely the following route A, if an unstable chromophore attaches under alkaline conditions. 
Thus, since there are typically many cases in which photofunctional molecules or other functional molecules are expensive, methods for synthesizing more pertinent functional PNA, namely methods for extremely rapidly introducing these functional molecules for (1) efficient introduction of functional molecules into a PNA backbone structure in the design of functional PNA monomer units, (2) synthesis routes in consideration of cost performance, and (3) adaptation to applications as gene diagnostic drugs, have been sought.
In consideration of the above problems, the inventors of the present invention found a novel method for producing functional PNA monomers consisting of synthesizing a photofunctional PNA monomer 4 nearly quantitatively by using a t-butoxycarbonylaminoethylamine derivative 6 for the PNA backbone structure, and condensing with an active ester form 5 containing the pentafluorophenyl group of 1 as indicated in the following route B. 
In addition, the inventors of the present invention found a different method for synthesizing functional PNA monomers by using a benzyloxycarbonyl-xcfx89-amino acid derivative instead of the above t-butoxycarbonylaminoethylamine derivative 6 for the PNA backbone structure (route C). Patent applications have already been made for these methods.
Thus, methods for ultimately synthesizing functional PNA are being established industrially that consist of synthesizing functional PNA monomers according to methods using either of the above routes B or C, followed by polymerization of those monomers. Namely, it is becoming possible to industrially synthesize large volumes of functional PNA used as PNA probes using existing functional PNA synthesis methods.
On the other hand, improvements are also being made on methods for producing functional PNA for the purpose of improving cost performance and allowing ultra-high-speed introduction of functional molecules. For example, a method has been reported in which functional molecules are introduced into PNA oligomers post-synthetically by using the following precursor PNA monomer unit as a different approach from the method described above using functional PNA monomer units (Oliver Seitz: Tetrahedron Letters 1999, 40, 4161-4164). 
In this method, after introducing the above precursor PNA monomer unit into a PNA oligomer, functional PNA is synthesized by additionally introducing a functional molecule.
However, this method has the disadvantage of there being limitations on the types of functional molecules that can be introduced.
For example, as indicated below, the commercially available photofunctional molecule, succinimide ester, is unable to be introduced. Although it is necessary to first introduce a linker such as Fmoc-Gly in order to introduce this photofunctional molecule, the above compound becomes difficult to use as a result of this. 
In addition, although DNA oligomers, RNA oligomers and PNA oligomers have been used in the past as fluorescent probes for introducing into cells, in order to introduce these into cells, they must naturally be able to pass through the cell membrane. However, since the surface of the cell membrane has a negative charge, it is extremely difficult to introduce DNA/RNA oligomers that are inherently negatively charged.
On the other hand, although PNA oligomers are electrically neutral, results have been obtained which indicate they are difficult in permeating the cell membrane. Thus, when introducing PIVA oligomers into a cell, that introduction must be facilitated by pretreating the membrane surface, or they must be introduced by using a transfection reagent.
However, in the case of introducing PNA oligomers by performing such treatment, even though the probe""s function may be demonstrated, there is ho guarantee that the behavior inherently demonstrated by the living body will always be accurately represented. Moreover, this is only true in the case of one cell, and in the case of numerous cells (individual body), their use is practically impossible.
On the basis of this current situation and viewpoint, the development of a fluorescent PNA probe having a membrane permeation function is considered to be useful.
It should be noted that fluorescent PNA probes having a membrane permeation function already exist. Examples include (1) a fluorescent PNA probe in which an oligopeptide having a membrane permeation function is linked to PNA, and (2) a fluorescent PNA probe in which a phospholipid having a membrane permeation function is linked to PNA. However, the portion of these probes other than the PNA is expected to be decomposed by enzymes such as proteases within cells after they have permeated the cell membrane, thereby causing them to be retained within the cell. Since this leads to excess PNA probes that were unable to capture the target losing their membrane permeation function and having difficulty in moving outside the cell in subsequent washing steps, this means that the gene expression system inherently possessed by the cell cannot be expressed accurately.
Thus, it is an object of the present invention to provide a novel method for synthesizing functional PNA having superior cost performance and which enables functional molecules to be introduced extremely rapidly, compounds used therein, and novel functional PNA.
As a result of extensive research in consideration of the above problems, the inventors of the present invention surprisingly found that, by optimizing the structure of precursor PNA monomer units, the above problems of the prior art can be overcome, and functional PNA can be synthesized over an extremely wide range, thereby leading to completion of the present invention.
More specifically, an aspect of the present invention relates to a method for producing a functional PNA oligomer, wherein a PNA monomer unit having adenine, guanine, cytosine or thymine protected by a protecting group is reacted with Fmoc-xcfx89-amino acid-BocPNA-OH represented by the following general formula (I): 
(wherein n represents an plus integer)
and after synthesizing PNA oligomer, a functional molecule having free carboxylic acid is introduced into that PNA oligomer followed by deprotecting of the protecting group.
Further, the present invention relates to the method described above, wherein the Fmoc-xcfx89-amino acid-BocPNA-OH is produced by a reaction between Fmoc-xcfx89-amino acid pentafluorophenyl ester and BocPNA-OH.
Further, the present invention relates to the method described above, wherein the Fmoc-xcfx89-amino acid pentafluorophenyl ester is produced by a reaction between Fmoc-xcfx89-amino acid and pentafluorophenol.
Further, the present invention relates to the method described above, wherein different functional molecule is introduced after introducing a functional molecule.
Still further, the present invention relates to the method described above, wherein the introduced functional molecule is chosen from a photofunctional molecule, a membrane-permeable functional molecule, an organ-selective functional molecule, a bactericidal functional molecule and a molecule-recognizing functional molecule.
Further, the present invention relates to the method described above, wherein the introduced functional molecule contains a photofunctional molecule and a membrane-permeable functional molecule.
In addition, the present invention relates to the method described above, wherein the photofunctional molecule is FITC, ROX, TAMRA or Dabcyl, and the membrane-permeable functional molecule is a water-soluble amino acid.
In addition, the present invention relates to the method described above, wherein the protecting group that protects adenine, guanine, cytosine or thymine is a Z group.
In addition, the present invention relates to the method described above, wherein synthesis of PNA oligomer contains condensation and elongation in a PNA chain using a solid-phase carrier for the tBoc method.
In addition, the present invention relates to the method described above, wherein the solid-phase carrier for the tBoc method is MBHA.
In addition, the present invention relates to the method described above, wherein introduction of a functional molecule having free carboxylic acid is carried out by dehydration condensation with a primary amino group obtained by selectively deprotecting the Fmoc group by piperidine treatment,
In addition, the present invention relates to the method described above, wherein Fmoc-xcfx89-amino acid-BocPNA-OH is a compound represented by the following general formula (I): 
(wherein n represents an integer of 1 through 15).
In addition, the present invention relates to the method described above, the method comprising one or more of the following steps a) through d) of:
a) reacting Fmoc-xcfx89-amino acid and pentafluorophenol in a step in which Fmoc-xcfx89-amino acid pentafluorophenylester is produced;
b) introducing Fmoc-xcfx89-amino acid into BocPNA-OH by reacting Fmoc-xcfx89-amino acid pentafluorophenyl ester with BocPNA-OH in a step in which Fmoc-xcfx89-amino acid-BocPNA-OH is produced;
c) producing PNA oligomer by reacting a PNA monomer unit with Fmoc-xcfx89-amino acid-BocPNA-OH in a step in which PNA oligomer is produced from Fmoc-xcfx89-amino acid-BocPNA-OH; and,
d) carrying out introduction of a functional molecule into PNA oligomer by dehydration condensation of a primary amino group obtained by selectively deprotecting an Fmoc group by piperidine treatment in a step in which a functional PNA oligomer is produced from the above PNA oligomer.
In addition, an aspect of the present invention relates to a compound represented by the following general formula (I): 
(wherein n represents an integer of 1 through 15).
In addition, the present invention relates to a method for producing the compound represented by general formula (I): 
(wherein n represents an integer of 1 through 15), the method comprising the introduction of Fmoc-xcfx89-amino acid by reacting Fmoc-xcfx89-amino acid with pentafluorophenol, and reacting that reaction product with BocPNA-OH.
In addition, the present invention relates to a compound represented by the following general formula (II): 
(wherein B""s each independently are the same or different and represent adenine, guanine, cytosine or thymine, R""s each independently are the same or different and represent an Fmoc group or a functional carboxylic acid derivative, R1 represents a hydrogen atom or a functional carboxylic acid derivative, a through h represent integers of 0 to 10, X1 through X3, Y1, Y2 and Z1 through Z5 all represent integers of 0 or more, X1+X2+X3xe2x89xa70, Y1+Y2 greater than 0, and Z1+Z2+Z3+Z4+Z5xe2x89xa70, provided that X1+X2+X3 and Z1+Z2+Z3+Z4+Z5 are not simultaneously 0, and in the case where X1+X2+X3=0, R1 is a functional carboxylic acid derivative).
In addition, the present invention relates to the compound described above, wherein Z1+Z2+Z3+Z4+Z5=0, and R1 is a hydrogen atom.
In addition, the present invention relates to the compound described above, wherein R includes a carboxylic acid derivative of methyl red.
In addition, the present invention relates to the compound described above, wherein X1+X2+X3=9, and Y1+Y2=1.
In addition, the present invention relates to the compound described above, wherein X1=3, X2=6 and Y1=1.
In addition, the present invention relates to the compound described above, wherein R or R1 represents a cell membrane-permeable functional molecule derivative.
In addition, the present invention relates to the compound described above, wherein R1 represents a functional carboxylic acid derivative.
In addition, the present invention relates to the compound described above, wherein X1=Z1=1.
In addition, the present invention relates to the compound described above, wherein Y1xe2x89xa72 and Z2=1.
In addition, the present invention relates to the compound described above, wherein a xe2x89xa66, bxe2x89xa64 and fxe2x89xa66.
In addition, the present invention relates to the compound described above, wherein R1 is a photofunctional carboxylic acid derivative.
Further, the present invention relates to a compound described above, represented by the following general formula (III): 
(wherein n represents an integer of 1 through 15).
In addition, the present invention relates to the method for producing the compound represented by the following general formula (III): 
(wherein, n represents an integer of 1 through 15), wherein Fmoc-xcfx89-amino acid is reacted with pentafluorophenol.
The present invention succeeds in being able to synthesize photofunctional PNA oligomers nearly quantitatively by introducing a precursor PNA monomer unit, in which Fmoc-xcfx89-amino acid has been introduced into a PNA backbone structure, namely Fmoc-xcfx89-amino acid-BocPNA-OH, into a PNA oligomer, followed by post-synthetically introducing a functional molecule.
According to the above characteristics, in the production method of the present invention, it is not necessary to use commercially available succinimide ester for the functional molecule to be introduced, but rather provided a compound has a carboxyl group, that compound can be used without problem and introduced quantitatively. Consequently, the production method according to the present invention has extremely superior cost performance.
In addition, by dividing the resin after introducing the precursor PNA monomer units into functional PNA oligomer, different functional molecules can be introduced into each resin. Thus, according to the production method of the present invention, an extremely rapid functional PNA oligomer synthesis procedure can be developed.
An example of a functional PNA oligomer that can be efficiently synthesized by the method of the present invention is the compound represented by the following general formula (II): 
(wherein B""s each independently are the same or different and represent adenine, guanine, cytosine or thymine, R""s each independently are the same or different and represent an Fmoc group or a functional carboxylic acid derivative, R1 represents a hydrogen atom or a functional carboxylic acid derivative, a through h represent integers of 0 to 10, X1 through X3, Y1, Y2 and Z1 through Z5 all represent integers of 0 or more, X1+X2+X3xe2x89xa70, Y1+Y2 greater than 0, and Z1+Z2+Z3+Z4+Z5xe2x89xa70, provided that X1+X2+X3 and Z1+Z2+Z3+Z4+Z5 are not simultaneously 0, and in the case where X1+X2+X3=0, R1 is a functional carboxylic acid derivative), wherein Z1+Z2+Z3+Z4+Z5=0 and R1 is a hydrogen atom.
According to the present invention, identical or different functional molecules can be introduced at a plurality of arbitrary sites in the compound represented by the above-mentioned general formula (II). Namely, although piperidine treatment and post-synthetic introduction of a functional molecule can be carried out collectively after introducing a PNA oligomer using the previously mentioned precursor PNA monomer units, this is indispensable in terms of rapidly designing antenna pedia that improve the cell membrane permeation function of PNA oligomers. The method according to the present invention is extremely superior with respect to this point as well.
An example of a compound produced in this manner is a compound in which Z1+Z2+Z3+Z4+Z5 greater than 0, R represents a cell membrane-permeable molecule derivative, and R1 represents a functional carboxylic acid derivative in the previously mentioned general formula (II).
This probe can be broadly divided into three regions consisting of fluorescent-labeled region, cell membrane permeation function region, and molecule-recognizing region, and has a form in which each of these are linked by means of linker sites (section represented by the suffixes of Z1 through Z5).
Both commercially available products as well as a novel fluorescent-labeled PNA monomer unit for which PCT application has already been filed by the inventors of the present invention may be-used for the fluorescent-labeled compound.
The molecule recognition site is synthesized using a commercially available PNA unit. This is characterized by the use of a novel PNA unit represented by general formula (I) for which a patent application has already been made in Japan for the membrane permeation function region. This novel PNA unit represented by general formula (I) is a precursor unit developed for post-synthetic introduction of functional molecules, and is characterized by allowing the collective introduction of molecules having the same function as was previously mentioned after introducing a plurality of these novel PNA units in a row.
Thus, according to the present invention, various functional molecules, without being limited to photofunctional molecules, can be both easily and extremely efficiently introduced into PNA.
Examples of such functional molecules include naphthalimide, flavin, dabcyl, biotin, FAM, rhodamine, TAMRA, ROX, HABA, pyrene and coumarine-type photofunctional monomer units, membrane-permeable functional molecules, organ-selective functional molecules, bactericidal functional molecules and molecule-recognizing functional molecules.
Namely, the term xe2x80x9cfunctionalxe2x80x9d in the present invention is not limited to photofunctionality, but also refers to all types of functions newly imparted to compounds by a certain modification, including membrane permeability, organ selectivity, bacteridical function and molecule recognition function.
Moreover, the term xe2x80x9cfunctional PNAxe2x80x9d in the present invention not only refers to the direct linkage of PNA monomers by a 2-(N-aminoethyl)glycine skeleton, but also to those containing a hydrocarbon chain and so forth in the form of a linker between them.
Other aspects and advantages of the invention will become apparent from the following description, taken in conjunction with the accompanying drawings, illustrating by way of example the principles of the invention.